Photosynthetic organisms that undergo oxygen-generating photosynthesis comprise 2 photochemical systems PSI and PSII. The photochemical reaction in PSII is initiated when chlorophyll a molecules in the PSII reaction center are excited and electrons are transferred to the initial electron acceptor (QA). In order to efficiently excite chlorophyll a molecules in the reaction center, a structure in which a chlorophyll a/b-protein complex referred to as an “antenna pigment” surrounds the reaction center and the antenna pigment efficiently transfers the captured light energy to the reaction center is formed on the thylakoid membrane. When the light is intensified, the number of photons that can be accepted by the antenna pigment per unit time is increased, and the photosynthetic rate is increased as a consequence. However, the number of electrons that can be accepted by QA per unit time is limited. Thus, the photosynthetic rate is substantially maximized at the light intensity referred to as a “light saturation point.” Even if the light intensity is further increased, the photosynthetic rate would not be increased. If the light intensity is further increased to a significant extent, the photosynthetic rate would rather be decreased. A decrease in the photosynthetic rate caused by the high-intensity light is referred to as “photoinhibition.”
Photoinhibition is often caused by a lowered PSII activity caused by a damaged D1 protein, which constitutes PSII. The D1 protein is damaged by light energy absorbed by manganese in the manganese cluster that also constitutes PSII. Such D1 protein damage is also observed under low-intensity light. Under low-intensity light, however, the damaged D1 protein is removed rapidly and replaced with a newly synthesized D1 protein. As a result of such rapid D1 protein repair, a PSII activity would not be lowered under low-intensity light. Under high-intensity light, however, an excess amount of light energy is absorbed by the antenna pigment, and an active oxygen species such as single oxygen is generated by the excessive reduction power. This active oxygen species inhibits novel synthesis of the D1 protein, the amount of the active D1 protein is decreased, and photoinhibition is then induced as a consequence (Takahashi, S., & Badger, M. R., 2011, Photoprotection in plants: a new light on photosystem II damage, Trends in plant science, 16 (1), 53-60).
In order to avoid generation of an excess amount of reduction power, photosynthetic organisms have a mechanism of converting excess light energy into heat energy (NPQ: non-photochemical quenching). In the case of Chlorophyta, the Viridiplantae (hereafter, referred to as “green algae”), a protein referred to as “LHCSR” has the shared responsibility of NPQ. LHCSR binds chlorophyll a/b to xanthophyll and it is in contact with the antenna pigment. The light energy absorbed by chlorophyll that is bound to LHCSR is transferred to xanthophyll, followed by thermal dissipation. LHCSR is also capable of thermal dissipation of the light energy accepted by the antenna pigment in the vicinity thereof. The C-terminus of LHCSR is exposed to the lumen inside the thylakoid membrane. When electron transfer is caused on the thylakoid membrane by the photosynthetic reaction, H+ migrates from the outside (the stroma) to the inside (the lumen) of the thylakoid membrane, H+ passes through ATP synthetase localized on the thylakoid membrane of the chloroplast, and it returns to the stroma while synthesizing ATP. When irradiated with high-intensity light, the amount of H+ introduced into the lumen upon photosynthetic electron transfer becomes larger than the amount of H+ discharged through the ATP synthetase, and a pH level of the lumen shifts toward a more acidic state. When the C-terminal amino acid sequence of LHCSR is exposed to a low pH state, LHCSR activity is enhanced, and efficiency of LHCSR-induced thermal dissipation is enhanced. In addition, the LHCSR gene is induced under high-intensity light, and the LHCSR content is increased. As such content is increased, the NPQ capacity is also increased. Since the LHCSR level is low and a pH level of the lumen is not low under low-intensity light, a majority of the light energy captured by the antenna pigment is transferred to the reaction center. Under high-intensity light, however, both the activity and the amount of LHCSR are increased, and a majority of the light energy captured by the antenna pigment would undergo thermal dissipation (Tokutsu, R., & Minagawa, J., 2013, Energy-dissipative supercomplex of photosystem II associated with LHCSR3 in Chlamydomonas reinhardtii, Proceedings of the National Academy of Sciences, 110 (24), 10016-10021).
In recent years, microalgae have drawn attention as raw materials of biomass fuels. Unlike land-dwelling creatures, microalgae live and grow in water. In an underwater region near the surface of water that is struck by sunlight in summer, microalgae also experience photoinhibition. However, photoinhibition occurs at different light intensities depending on microalgae species (Singh, S. P., & Singh, P., 2015, Effect of temperature and light on the growth of algae species: A review, Renewable and Sustainable Energy Reviews, 50, 431-444).
Such photoinhibition damages microalgae when they are cultured outdoors. When chlorella was cultured in a very shallow culture pool outdoors, for example, growth inhibition was particularly significant when the chlorella cell density was low. When the chlorella cell density is low, many chlorella cells receive sunlight directly from the sun, and photoinhibition leads to growth inhibition as a consequence (Masojidek, J., Kopecky, J., Giannelli, L., & Torzillo, G., 2011, Productivity correlated to photobiochemical performance of Chlorella mass cultures grown outdoors in thin-layer cascades, Journal of industrial microbiology & biotechnology, 38 (2), 307-317).
The Pseudochoricystis ellipsoidea (P. ellipsoidea) Obi strain, which is an unicellular green algae belonging to the class Trebouxiophyceae (hereafter, it is referred to as the “Obi strain”), can grow at a pH level of 3.5 or lower and such strain can be cultured in an open culture system disclosed in JP Patent Publication (Kokai) No. 2014-117202 A. Thus, studies concerning the use thereof for biomass fuel production have been in progress (Kasai, Y., Oshima, K., Ikeda, F., Abe, J., Yoshimitsu, Y., & Harayama, S., 2015, Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea, Biotechnology for biofuels, 8 (1), 1-12; and Matsuwaki, I., Harayama, S., & Kato, M., 2015; Assessment of the biological invasion risks associated with a massive outdoor cultivation of the green alga, Pseudochoricystis ellipsoidea. Algal Research, 9, 1-7). When the Obi strain was cultured in outdoor raceway culture equipment in summer, however, strong growth inhibition was observed at a low cell density as described with regard to the chlorella cells.
As a solution to the problem resulting from such photoinhibition, a mutant that is resistant to photoinhibition may be separated. There was one report concerning separation of photoinhibition-resistant microalgae in the past. After the Chlamydomonas reinhardtii cells were irradiated with UV to induce mutagenesis, mutants that would grow under high-intensity light (2,500 μmol photons m−2 s−1) were selected, and 2 strains were separated. Both mutants had mutations in the gene with the gene ID of Cre02.g085050. Thus, this gene was designated as the putative light response signaling protein 1 (LRS1). LRS1 comprised evolutionary conserved domain sequences; i.e., the RING domain in the N-terminal amino acid sequence and the WD 40 domain in the C-terminal amino acid sequence. An example of a protein comprising such 2 domains is the COP1 protein existing in Arabidopsis thaliana. When the COP1 amino acid was compared with the LRS1 amino acid sequence, the degree of sequence homology was found to be high in the N-terminal and C-terminal domains, although significant homology was not observed in the central region (Schierenbeck, L., Ries, D., Rogge, K., Grewe, S., Weisshaar, B., & Kruse, O., 2015, Fast forward genetics to identify mutations causing a high light tolerant phenotype in Chlamydomonas reinhardtii by whole-genome-sequencing, BMC genomics, 16 (1), 57). In Arabidopsis thaliana, COP1 forms a complex with a protein referred to as “SPA1,” and it forms an ubiquitin transferase (E3 ubiquitin ligase) together with a protein such as CUL4, RBX1, or DDB1. SPA1 comprises a kinase domain at the N-terminus and the WD40 domain at the C-terminus. The COP1 WD40 domain is considered to recognize the target protein of the ubiquitin transferase in combination with the SPA1 WD40 domain (Biedermann, S., & Hellmann, H., 2011, WD40 and CUL4-based E3 ligases: lubricating all aspects of life, Trends in plant science, 16 (1), 38-46). SPA1 is a protein constituting an ubiquitin transferase, SPA1 itself is ubiquitinated by many optical signals, and it is degraded by a proteasome (Chen, S., Lory, N., Stauber, J., & Hoecker, U., 2015, Photoreceptor Specificity in the Light-Induced and COP1-Mediated Rapid Degradation of the Repressor of Photomorphogenesis SPA2 in Arabidopsis, PLoS Genet, 11 (9), e1005516). Specifically, activity of the COP1/SPA1 complex of Arabidopsis thaliana is associated with transmission of many optical signals through regulation of its own activity. It is thus deduced that LRS1 of Chlamydomonas is also a protein associated with transmission of high-intensity light stress signals.